Exendin-4 is a natural product isolated and identified from the saliva of the Gila monster (Heloderma suspectum). It is a 39-amino acid peptide, whose sequence is His1-Gly-Glu-Gly-Thr5-Phe-Thr-Ser-Asp-Leu10-Ser-Lys-Gln-Met-Glu15-Glu-Glu-Ala-Val-Arg20-Leu-Phe-Ile-Glu-Trp25-Leu-Lys-Asn-Gly-Gly30-Pro-Ser-Ser-Gly-Ala35-Pro-Pro-Pro-Ser39-NH2 (SEQ ID NO: 1). Exendin-4 is an analog of glucagon-like peptide-1 (GLP-1). It is a GLP-1 receptor agonist which can activate GLP-1 receptor, and has biological activities such as the activities of controlling glyceride, promoting insulin secretion, reducing circulating glucagons, enhancing pancreatic β-cell quality, inhibiting gastric emptying, reducing intake of nutrition, and increasing sensitivity to insulin. Exendin-4 can simulate the hormones secreted in vivo naturally and therefore help regulate blood sugar level in human bodies. Moreover, it would stop exerting the role when it is not needed in patients. Another characteristic of Exendin-4 lies in the recovery of the first phase of secretion of β cells responsible for insulin secretion. The first phase of secretion refers to insulin secretion induced by food intake. However, patients with Type II diabetes lose the ability to raise such a response early in the course of the disease. Exendin-4 can not only reduce blood sugar, but also enhance the losing cellular functions, and thus is a great progress for the treatment of Type II diabetes.
Exendin-4 is approved by US FDA in April, 2005 for improving glycemic control in patients with Type II diabetes, when glycemic control is not ideal by using dimethyldiguanide and sulfonylurea drugs. It is shown in clinical results that Exendin-4 has a significant therapeutic effect on the treatment of diabetes and has small side-effect, but brings about inconvenience to patients due to two subcutaneous administrations per day.
Due to the efficacy and the unique blood glucose-dependent mechanism of Exendin-4, it is more suitable for the preparation of a sustained-release formulation. The sustained-release injection Exendin-4 LAR, a product developed by Amylin Co. co-operated with Alkennes Co. in May, 2000, has finished phase II clinical trial, and the results show that after 15 weeks, the medicament in each of the two dose levels was well tolerated, and both the blood concentrations of Exendin-4 reached the therapeutic range as expected. Meanwhile, since Exendin-4 has a significant advantage in terms of the therapeutic mechanism of Type II diabetes, the development of its long-acting analogs also becomes a research and development hotspot in many foreign pharmaceutical companies.
Studies show that polypeptide drugs can still maintain good bioactivity after modified by polyethylene glycol (PEG), and such modification can significantly prolong in vivo half-life in organisms. Currently, there are reports on PEG-modification of Exendin-4, in which the products having PEG modified at the amino group of His1, Lys12 and Lys27, respectively, are provided. In the research on metabolism in rats, it was found that their metabolic half-life was much longer than Exendin-4 (Jin Zhou et al., Eur. J. Pharma. Biopharm., 2009, 72:412-417). In addition, it was also reported that when Exendin-4 was modified by fatty acid, lithocholic acid or hyaluronic acid at ε-amino group of Lys12 and Lys27, single modified and double modified compounds were obtained, respectively (Su Young Chae et al., J. Control. Release, 2010, 144(1): 10-16; J. Control. Release, 2010, 142:206-213; Biomaterials, 2010, 31:4121-4128).
However, as understood by a person skilled in the art, no matter Exendin-4 is modified by PEG, or by fatty acid, lithocholic acid or hyaluronic acid, there is a problem concerning poor specificity to the modification sites. Thus, the product resulted from the modification might be a mixture of different compounds modified at different single sites, which is quite disadvantageous for the subsequent pharmaceutical application.
Therefore, there is still need in the art to provide a method for site-directed modification of Exendin-4 whilst maintaining the biological activity of Exendin-4, and provide the modified Exendin-4, such as PEG-modified Exendin-4, in order to address the problem concerning poor specificity to modification of Exendin-4, such as PEG-modification of Exendin-4.